Positive selection of self-MHC-reactive T cells by individual peptide-MHC class II complexes -- Data Supplement - Supporting Methods -- Proceedings of the National Academy of Sciences

Supporting information for Barton et al. (2002) Proc. Natl. Acad. Sci. USA 99 (10), 6937–6942. (10.1073/pnas.102645699)

Methods
Blocking of Ea–A^b and CLIP–A^b Complexes in Vivo.
To block Ea–A^b and CLIP–A^b complexes, Ea ´ CLIP mice were injected i.p. on the day of birth and every other day thereafter with 200 m g per mouse of YAe (IgG2b) mAb or a 1:1 mixture of 15G4 (IgG1) and 30-2 (IgG2a) mAb specific for Ea–A^b and CLIP–A^bcomplexes, respectively. As a control, mice were treated similarly with normal mouse IgG. After 3–3.5 weeks of treatment, splenocytes were isolated and cultured in triplicates for 96 h at 4 ´ 10⁵ cells per well in flat-bottom 96-well plates with titrated numbers of irradiated syngeneic or B6 splenocytes (2,000 rad). Each well was pulsed with 0.5 mCi (1 Ci = 37 GBq) [³H]thymidine after 72 h and harvested after 96 h. To confirm that splenic cell populations isolated from antibody-treated mice contained comparable numbers of CD4 T cells, splenocytes were stained for CD4, CD8, and T cell antigen receptor-b chain and analyzed by flow cytometry as described above. Analysis of MHC-Bound Peptides.
To analyze the repertoire of MHC class II-bound peptide in human invariant chain (Ii)-peptide transgenic mice we purified A^b molecules from thymi, isolated the associated peptides, and determined their mass by microcolumn HPLC and electrospray ionization mass spectrometry (1). Mass profiles of the peptide repertoires were created by averaging the scans across the range of peptide elution. Peptide elution traces were very reproducible and allowed an identical number of scans to be averaged for all experiments. Peptides profiles of A^b purified from Ea-Ii⁰, Ea-dbl⁰, and H-2M⁰ thymi are shown in Fig. 6. The ion chromatograms from each peptide preparation were dominated by masses that corresponded to a set of peptides with multiple charge states. Most of the prominent masses matched precisely to peptides containing the core Ea or class II-associated invariant chain peptide (CLIP) sequence and displayed the "ragged ends" typical of MHC class II-bound peptides. Ea sequences frequently were flanked by human Ii residues. In a few instances, we were unable to assign a peptide sequence to an observed mass, which most likely is because of our inability to identify the amino acid modifications and/or adducts responsible for the observed mass. Potential low-level peptide signals other than the predominant peptides are impossible to extract because of chemical noise inherent to the current technology. It is clear from the data, though, that almost all the prominent masses correspond to expected peptides for each mouse genotype. Analogous results were obtained in an analysis of CD22-dbl⁰ thymi (data not shown). Importantly, the same peaks of Ii-Ea nested peptides were seen in both the Ea-Ii⁰ and Ea-dbl⁰ chromatograms. Similarly, CLIP peptide variants isolated from H-2M⁰ thymi were not different from those we routinely find in A^b isolated from wild-type thymi or splenocytes. These results suggest that the presence or absence of H-2M does not affect the predominant peptides found in Ii-peptide mice. Furthermore, the different Ii-peptide proteins are processed similarly.

1. McCormack, A. L., Eng, J. K., DeRoos, P. C., Rudensky, A. Y. & Yates, J. R. (1996) in Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, ed. Synder, A. P. (Am. Chem. Soc., Washington, DC), pp. 207–225.